Several New World arenavirus (NWA) cause hemorrhagic fever (HF) diseases in humans that are characterized by high morbidity and mortality. Moreover, because of their stability, high morbidity and potential for aerosol transmission, HF-causing NWA represent credible biodefense threats.Concerns posed by human pathogenic NWA are aggravated by the lack of FDA-licensed vaccines and current anti-arenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. The significance of HF-causing NWA in human health and biodefense readiness, together with the limited existing armamentarium to combat them, underscore the importance of developing effective countermeasures to combat NWA infections of humans. For several viruses broadly neutralizing antibodies (BNAb) have been isolated from individuals who recovered from infection and shown to bind to highly conserve epitopes within viral surface glycoproteins (GP) and capable of protecting against viruses with high degree of antigenic variability. Therefore, BNAb provide a powerful tool for antiviral immunotherapy. Moreover, information regarding the interaction of BNAb with their highly conserved epitopes and their mechanisms of neutralization can facilitate the design of novel universal vaccines and the discovery of broad-spectrum antiviral drugs. In this exploratory R21 application we propose first to generate cross-reactive variable (V) region humanized monoclonal antibodies (hVMAbs) against NWA surface GPs using sequential plasmid DNA immunization of VelocImmune mice with GPs from the genetically distantly related NWA (Aim 1). We will characterize these hVMAbs regarding their breadth of reactivity against GPs of representative members from all three NWA clades (Aim 2), including all known HF-causing NWA. We will select hVMAbs exhibiting broad NWA cross-reactivity to identify those with broadly neutralizing activity (BNhVMAbs) against NWA in cell-based assays (Aim 3) and to initially assess their in vivo therapeutic activity using infection of the mouse with recombinant LCM viruses expressing NWA HF-causing GPs of interest (Aim 4). The identification and characterization of NWA GP-specific BNhVMAbs will represent a first and necessary step for the development of an antibody (Ab)-based therapy against HF-causing NWA. Furthermore, characterization of the epitopes recognized by identified NWA GP-specific BNhVMAbs will facilitate the generation of immunogens able to induce similar broadly neutralizing (BN) responses to generate a polyvalent NWA vaccine, as well as the rationale design of broad-spectrum anti-NWA antiviral drugs.